Petunia plants having a unique flower color pattern

ABSTRACT

Petunia  plants comprising at least one dominant allele conferring a novel spotted flower color pattern are disclosed. Also disclosed are methods of breeding with the  Petunia  plants having the novel spotted flower color pattern to provide new, distinct and stable  Petunia  plants having flowers with a unique spotted flower color pattern. This spotted flower color pattern has not been observed in any  Petunia  known species.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 62/316,271 filed on Mar. 31, 2016, the contents of which are herebyincorporated in their entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and is hereby incorporated by referenceinto the specification in its entirety.

BACKGROUND

All publications cited in this application are herein incorporated byreference. The present disclosure relates to the field of ornamentalPetunia plants and plant breeding. The disclosure provides new, distinctand stable Petunia plants having inflorescences with a unique spottedflower color pattern. This color pattern has not been observed in anyknown Petunia, but was discovered through the Applicant's breedingprogram.

The genus Petunia belongs to the plant family Solanaceae and is dividedinto 14 different species, which are endemic to South America. Thegeographical distribution includes temperate and subtropical regions ofArgentina, Uruguay, Bolivia, and Brazil, with a center ofdiversification in southern Brazil. The common garden Petunia,Petunia×hybrida (Hook.) Vilm, now commonly known as Petunia hybrid, isderived from a cross of the two species P. integrifolia and P.axillaris. The garden Petunia was first obtained through hybridizationin 1834 by Atkins of Northhampton, a British nurseryman, and it soonspread to all European gardens (Sink, K. C. Ed. (1984) In: Petunia:Monographs on Theoretical and Applied Genetics 9. Springer-Verlag,Berlin, pp. 3-9). Today, it is used worldwide as an outdoor pot plant,in window boxes or as a bedding plant and is one of the most importantornamental plants. In 1983, Wijsman showed that crosses between furtherPetunia species as well as between the garden Petunia and other speciesare possible ((Wijsman, H. J. W. (1983). On the interrelationships ofcertain species of Petunia. II. Experimental data: Crosses betweendifferent taxa. Acta Bot. Neerl. 32:97-107; Wijsman, H. J. W. and JongJ. H. (1985) On the interrelationships of certain species of Petunia.IV. Hybridization and nomenclatural consequences in the Petunia group.Acta Bot. Neerl. 34: 337-349). Therefore, it is probable that furtherPetunia species have contributed to the modern varieties.

In commercial Petunia breeding, there has always been big interest inthe development of new flower colors and color patterns, the modernPetunia varieties today cover a broad range of different flower colorsas well as combinations therefrom. The flower color of Petunia isdetermined mainly by flavonoids, whereas the related species Calibrachoauses anthocyanidins as well as carotenoids for the red flower color(Murakami, Y., Fukui, Y. Watanabe, H., Kokubun, H., Toya, Y. and T. Ando(2004) Floral coloration and pigmentation in Calibrachoa cultivars.Journal of Horticultural Science & Biotechnology, 79(1): 47-53). Thebasic chromosome number of the genus Petunia is seven; by thischaracteristic, as well as by further morphological traits, Petunia canbe differentiated from Calibrachoa (Joao Renato Stehmann, Aline P.Lorenz-Lemke, Loreta B. Freitas and Joao Semir (2000) The Genus Petunia.In: T. Gerats and J. Strommer (Eds.), Petunia—Evolutionary,Developmental and Physiological Genetics. Second edition, SpringerVerlag, Berlin, pp. 1-28), but the two genera can be intercrossed.

Petunia can be propagated from seed, cuttings, and tissue culture.Germination, cutting propagation, and tissue culture protocols forPetunia are well-known in the art.

Petunia is an important and valuable ornamental plant. Thus, acontinuing goal of ornamental plant breeders is to develop plants withnovel characteristics, such as color, growth habit, and hardiness. Toaccomplish this goal, the breeder must select and develop plants thathave traits that result in superior Petunia varieties.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated herein and form a partof the specification, illustrate some, but not the only or exclusive,example embodiments and/or features. It is intended that the embodimentsand figures disclosed herein are to be considered illustrative ratherthan limiting.

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the Patent Office upon request and payment of thenecessary fee.

FIG. 1 is a photo of the Petunia plant commercially designated‘KLEPH15313’ showing the novel spotted flower color pattern, disclosedin the present application. The novel spotted flower color patternconsists of white spots on a purple base flower color.

FIG. 2 is a photo showing a close-up of an individual flower of theplant shown in FIG. 1.

FIG. 3 is a photo showing a close-up of an individual flower ofproprietary Petunia line ‘PH-2015-1074’ showing white spots on a redpetal color.

FIG. 4 is a photo showing a close-up of an individual flower ofproprietary Petunia line ‘PH-2015-1065’ showing yellow-green spots incombination with a yellow-green star pattern on a base red-purple petalcolor

FIG. 5 is a photo showing a close-up of an individual flower ofproprietary Petunia line ‘PH-2016-2150’ showing flowers having acream-yellow spotted flower color pattern in combination with acream-yellow star pattern on a dark violet base flower color. This photois an example of the spotted flower color pattern in combination with aborder pattern and a star pattern.

FIG. 6 is a photo showing a close-up of an individual flower ofproprietary Petunia line ‘PH-2015-1874’ showing white spots incombination with a white border on a blue base petal color. This photois an example of a flower with a solid border pattern in combinationwith the spotted flower color pattern of the present application.

FIG. 7 is a photo showing a close-up of an individual flower ofproprietary Petunia line ‘PH-2016-2068’ showing white spots on red starpattern in combination with a white base petal color.

FIG. 8 is a photo showing a close-up of an individual flower ofproprietary Petunia line ‘PH-2015-1080’ showing white spots on a pinkbase petal color.

FIGS. 9A-9E show that flower coloring of Petunia×hybrida variety‘KLEPH15313’ infected with CMV can lead to changes in flower coloring.Flowers of this variety are originally purple with either white dots (A)or white petal tips and dots (B), which can be altered in infectedplants to contain a purple smear in the white petal tips with vanishingwhite dots (C) and even to completely purple flowers (D). Only the clonePV-0036 also led to leaf symptoms with dark green dots on light greenleaves (E).

FIGS. 10A1-10C4 show flower coloring of Petunia×hybrida lines showingdifferent flower color patterns with and without CMV infection: line‘PH-2014-0028’ uninfected (10A1 and 10A2) and infected (10A3 and 10A4);line ‘PH-2007-0103’ uninfected (10B1 and 10B2) and infected (10B3 and10B4); line ‘PH-2015-1874’ infected (10C1 and 10C2) and uninfected (10C3and 10C4).

FIG. 11 shows the expression analysis of Chalcone Synthase A gene(CHS-A) from RNA of white and colored petal areas of Petunia×hybridavariety ‘KLEPH15313’ and Picotee-patterned line ‘PH-2007-0103’.Expression of the actin gene was used as internal control.

SUMMARY

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope. In various embodiments, one or more ofthe above-described problems have been reduced or eliminated, whileother embodiments are directed to other improvements.

According to one embodiment, there is provided a Petunia plantcomprising at least one dominant allele that produces a spotted flowercolor pattern, wherein said spotted flower color pattern compriseshaving one or more spots on at least one petal, wherein a sample ofrepresentative seed of said Petunia plant comprising at least onedominant allele that produces a spotted flower color pattern. A furtherembodiment relates to said Petunia plant having said spotted flowercolor pattern that was deposited under NCIMB No. 42563. A furtherembodiment relates to wherein said spots may be white, cream, yellow,yellow-green, or combinations thereof in color. A further embodimentrelates to wherein said spots may be circular or irregular in shape andmay also vary in size. A further embodiment relates to wherein saidPetunia plant showing said spotted color pattern may have a flower colorof yellow, orange, red, brown, blue, black, pink, violet, orcombinations and shades thereof. A further embodiment relates to whereinsaid Petunia plant having said spotted flower color pattern is incombination with a different flower pattern comprising a star pattern, aborder pattern, or a star pattern and a border pattern. A furtherembodiment relates to wherein said Petunia plant showing having saidspotted flower color pattern has a mature flower size between 2.5 cm and10.5 cm.

Another embodiment relates to tissue culture produced from tissue,callus, or cells from the Petunia plants disclosed in the subjectapplication, wherein said tissue, callus, or cells are produced from aplant part selected from the group consisting of pollen, ovules,embryos, protoplasts, meristematic cells, callus, leaves, anthers,cotyledons, hypocotyl, pistils, roots, root tips, flowers, seeds,petiole, and stems.

Another embodiment relates to a method for producing Petunia seed, saidmethod comprising crossing two Petunia plants and harvesting theresultant Petunia seed, wherein at least one Petunia plant is a Petuniaplant having the spotted flower color pattern. A further embodimentrelates to planting and growing the seed from said above cross toproduce a Petunia plant having the spotted flower color pattern.

Another embodiment relates to a method of vegetatively propagating aPetunia plant of in the instant application comprising the steps of: (a)collecting tissue capable of being propagated from the plant accordingto a Petunia plant of in the instant application; (b) cultivating saidtissue to obtain proliferated shoots; and (c) rooting said proliferatedshoots to obtain rooted plantlets.

Another embodiment relates to a Petunia plant having a novel spottedflower color pattern, wherein said spotted flower color patterncomprises a Petunia plant having at least one flower having one or morespots on at least one petal, and wherein said pattern is produced from apost-transcriptional gene silencing of the Chalcone Synthase A gene insingle randomly distributed cells of the petals. A further embodimentrelates to wherein said spots may be white, cream, yellow, yellow-green,or combinations thereof in color. A further embodiment relates towherein said spots may be circular or irregular in shape and may alsovary in size. A further embodiment relates to wherein said Petunia plantshowing said spotted color pattern may have a flower color of yellow,orange, red, brown, blue, black, pink, violet, or combinations andshades thereof. A further embodiment relates to wherein said Petuniaplant having said spotted flower color pattern has in combination with adifferent flower pattern comprising a star pattern, a border pattern, ora star pattern and a border pattern. A further embodiment relates towherein said Petunia plant having said spotted flower color pattern hasa mature flower size between 2.5 cm and 10.5 cm.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

SEQ ID NO:1 discloses the Petunia×hybrida primer CHSA-for (forward).

SEQ ID NO:2 discloses the Petunia×hybrida primer CHSA-rev (reverse).

Definitions

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. Allele is any of one or more alternative forms for a gene.

Border Pattern. A “border pattern” refers to a pattern expressed on theflower where the color along the outer margin of the flower is differentfrom the color of the rest of the flower. The color of the border alongthe outer margin of the flower may be thick or thin and solid orsemi-solid. The color of the border along the outer margin and the restof the flower may also vary in all colors and border patterns. Color, asreferred to here, includes all pigmented colors and shades in-between,white, and unpigmented.

Gene. As used herein, “gene” refers to a segment of nucleic acid.

Locus. A locus is the position or location of a gene on a chromosome.

Progeny. As used herein, the descendants of one or more of the parentallines and includes an F₁ Petunia plant produced from the cross of twoPetunia plants where at least one plant includes a Petunia plantdisclosed herein and progeny further includes, but is not limited to,subsequent F₂, F₃, F₄, F₅, F₆, F₇, F₈, F₉, and F₁₀ generational crosseswith the recurrent parental line.

RHS. RHS refers to the acronym for Royal Horticultural Society thatpublishes a color chart used in the plant industry. All RHS colorsreferred to herein are from the RHS 2007 edition.

Spotted Flower Color Pattern. As used herein, “spotted flower colorpattern” refers to a pattern expressed as one or more spots on at leastone petal of the flower. The spots range in shape from circular toirregular, vary in size, and vary in color from white, yellow,yellow-green, cream and combinations thereof.

Star Pattern. A “star pattern” refers to a pattern expressed on theflower where streaks of color longitudinally divide the pigmentedsections of the flower. The streaks are sometimes referred to as linesand may be thin or thick and solid or semi-solid. The streaks or linesradiate from the center or approximately the center of the flowertowards the outer margin of the flower. The streaks or lines may radiateall the way to the outer margin of the flower or partially towards theouter margin of the flower. The streaks or lines may vary in color andcolor patterns.

DETAILED DESCRIPTION

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine improved combination ofdesirable traits from the parental germplasm. These important traits mayinclude flower color, certain plant characteristics, higher vigor,resistance to diseases and insects, better stems and roots, tolerance todrought and heat, and better commercial plant and flower quality.

According to one embodiment, there is provided a Petunia plantcomprising at least one dominant allele that produces a spotted flowercolor pattern, wherein said spotted flower color pattern compriseshaving one or more spots on at least one petal, wherein a sample ofrepresentative seed of said Petunia plant comprising at least onedominant allele that produces a spotted flower color pattern isdeposited under NCIMB No. 42563. A further embodiment relates to saidPetunia plant and wherein said spots may be white, cream, yellow,yellow-green, or combinations thereof in color. A further embodimentrelates to wherein said spots may be circular or irregular in shape andmay also vary in size. A further embodiment relates to said Petuniaplant and wherein said Petunia plant showing said spotted color patternmay have a flower color of yellow, orange, red, brown, blue, black,pink, violet, or combinations and shades thereof. A further embodimentrelates to said Petunia plant and wherein said Petunia plant having saidspotted flower color pattern has in combination with a different flowerpattern comprising a star pattern or a border pattern. A furtherembodiment relates to said Petunia plant and wherein said Petunia planthaving said spotted flower color pattern has a mature flower sizebetween 2.5 cm and 10.5 cm.

The unique spotted flower color pattern comprises spots of a differentcolor than the basic color of the respective flower.

A further embodiment relates to a Petunia plant comprising at least onedominant allele that produces a spotted flower color pattern, whereinsaid spotted flower color pattern comprises a Petunia plant having atleast one flower having one or more spots on at least one petal, whereina sample of representative seed of said Petunia plant comprising said atleast one dominant allele that produces a spotted flower color patternis deposited under NCIMB No. 42563.

The Petunia plants having a spotted flower color pattern disclosed inthe present application have shown uniformity and stability, asdescribed in the following section. The Petunia plants having a spottedflower color pattern disclosed in the present application have beenasexually and sexually reproduced a sufficient number of generationswith careful attention to uniformity of plant type and has beenincreased with continued observation for uniformity.

Origin of the Spotted Flower Color Pattern in Petunia

The unique spotted flower color pattern on Petunia flowers is the resultof the Applicant's professional breeding program.

In 2010, a first cross was made between the P. grandiflorum seed variety‘Can Can’ with the garden Petunia variety ‘Veranda Navy Blue’. Further,a second cross was made between the variety ‘Can Can’ with theproprietary variety FAMOUS ‘Dark Blue’ (‘KLEPH08153’). The varietiesFAMOUS ‘Dark Blue’ and ‘Veranda Navy Blue’ both were solid-colored bluein flower color, whereas the flowers of the variety ‘Can Can’ showed awhite center on a burgundy basic flower color. From the resultingprogenies of each cross, Applicant selected two seedlings, one from eachcross, and designated the seedlings ‘PH-2011-2636’ and ‘PH-2011-2629’.Both seedlings displayed flower colors having a white center and apurple border. ‘PH-2011-2636’ and ‘PH-2011-2629’ were crossed in 2011,and in 2012, a single seedling, ‘PH-2012-3528’ was selected from theprogeny population of that cross, which was the first seedling showingthe novel spotted flower color pattern.

EXAMPLES

1. Genetic Background and Segregation Pattern

From 2012-2016, 155 crosses involving the spotted flower color patternwere performed in total, from which 25,248 seedlings were germinated andevaluated (please see Table 1). In Table 1 below, column one shows theyear that the crosses were conducted, column two shows the total numberof crosses conducted during that year, column three shows the totalnumber of potted seedlings as a result of all of the crosses conductedduring that year, column four shows the average number of progeny percross (column 3 number divided by column 2 number), column five showsthe minimum number of progeny observed from a given cross for that year,and column six shows the maximum number of progeny observed from a givencross that year.

Data in Table 1 comprises only those progenies, of which at least oneparent expressed the spotted color flower pattern. The progeniescomprised on average 163 seedlings, whereas the number of the individualprogenies varied from minimum of 1 to 493 seedlings (please see Table1). Among the 155 individual crosses, not all of the seedlings from eachcross showed the novel spotted flower color pattern, but everypopulation showed at least one spotted seedling. Based on thisobservation, it is postulated that the inheritance of the novel Petuniaspotted flower color pattern is dominant. However, the segregationpatterns indicate that the dominance is incomplete and potentially, morethan one gene is involved.

TABLE 1 Number of crosses with plants showing the novel spotted colorpattern and population size of the resulting progenies Potted Min. Max.Crosses seedlings Average number of number of (total (total number ofprogeny progeny Year number) number) progeny observed observed 2012 91590 177 75 208 2013 9 660 73 30 90 2014 35 10986 314 4 493 2015 36 6404178 2 397 2016 66 5608 85 1 208 Total 155 25248 163

Table 2 shows an overview of the proprietary breeding lines andcommercial varieties which were used as parental lines in crosses toproduce the seedling progenies in Table 1. The novel spotted flowercolor pattern was bred into a broad range of different flower colors,flower shapes, flower sizes, and growing habits. A number of bothproprietary and commercial varieties were used as parental lines. Linesin column one indicate those lines proprietary to Applicant; those linesin column two third-party commercial lines.

TABLE 2 Parental lines crossed with spotted Petunia parental line toproduce spotted Petunia progeny Applicant/proprietary variety Thirdparty commercial variety used as crossing parent used as crossing parentALPE TUNIA ‘Dark Blue’ ‘Sunshine Ray’ BONNIE ‘Magenta’ POTUNIA ‘CobaltBlue 2016’ BONNIE ‘Red ′14’ POTUNIA ‘Red 2016’ FAMOUS ‘Blue’ SURPRISE‘Marine’ FAMOUS ‘Dark Violet Picotee’ SURPRISE ‘Midnight Blue’ FAMOUS‘Electric Orange’ SURPRISE ‘Midnight Cowboy’ FAMOUS ‘Electric Purple’Sweetunia ‘Hot Rod Red’ FAMOUS ‘Purple evol.’ ‘Viva Bright Red’ FAMOUS‘Red Fire ′14’ ALPE TUNIA ‘Dark Purple’ ‘Lilac Picotee’ SURFINIA ‘EarlyLight Yellow’ ‘PH-2011-2416’ SURFINIA ‘Early Pat Red’ ‘PH-2012-306’SURFINIA ‘Purple’ ‘PH-2013-4154’ SURFINIA ‘Star Deep Violet’‘PH-2013-4155’ SURFINIA ‘Star Peach’ ‘PH-2013-4258’ SURFINIA ‘StarPurple’ ‘PH-2013-4273’ SURFINIA ‘Star Rose’ ‘PH-2013-4277’ SURFINIA‘Star Violet’ ‘PH-2013-4373’ SURFINIA ‘Table Dark Red’ ‘PH-2014-0004’SURFINIA ‘Violet’ ‘PH-2014-0035’ SURFINIA ‘Violet 2006’ ‘PH-2014-0237’SANGUNA ‘Patio Red’ ‘PH-2014-0594’ SANGUNA ‘Purple Edge’ ‘PH-2014-0756’SANGUNA ‘Purple Picotee’ ‘PH-2014-0797’ ‘PH-2014-1019’ ‘PH-2015-1153’2. Breeding of ‘KLEPH15313’ (Also Commercially Known as ‘KLEPH15313’Petunia, (FIGS. 1 and 2)

Petunia line ‘KLEPH15313’ is the result of a planned breeding program.‘KLEPH15313’ was selected from a cross between ‘PH-2012-3528’, which wasthe first seedling discovered by Applicant showing the novel spottedflower color pattern and the variety ‘Duesurmar’, which hassolid-colored blue flowers. The variety ‘KLEPH15313’ is amedium-compact, semi-spreading and cold tolerant Petunia plant, whichdisplays the unique spotted flower color pattern. In FIG. 1, a wholeplant of ‘KLEPH15313’ is depicted showing the spotted flower colorpattern on all of the flowers of the plant. FIG. 2 depicts a close-up ofa flower from FIG. 1 showing the spotted flower color pattern. In thisexample, the spotted flower color pattern shown is white spots on apurple base flower color. The spots vary from circular to irregularshape, vary in size, and are present on at least one petal of theflower.

3. Breeding of the Line ‘PH-2015-1074’ (FIG. 3)

Petunia line ‘PH-2015-1074’ displays petals having the spotted flowercolor pattern of white spots on a red base flower color, and is theresult of a planned breeding program. In 2012, ‘PH-2012-3528’, which wasthe first seedling discovered by Applicant showing the novel spottedflower color pattern, was crossed with a proprietary Petunia seedlingdesignated ‘PH-2011-2416’, which had solid-red colored flowers. Theresulting progeny from this cross, ‘PH-2013-4407’, displayed purpleflowers with a white spotted flower color pattern. ‘PH-2013-4407’ wasfurther crossed with a solid black colored commercial variety named‘Surprise Midnight Cowboy’ (‘Duesurmidco’). Among the progeny of thislast cross, a single seedling designated ‘PH-2014-0009’ was selectedwhich displayed purple flowers with a yellow spotted flower colorpattern. A further cross of ‘PH-2014-0009’ with a proprietary Petuniaseedling designated ‘PH-2014-0797’ that does not have the spotted flowercolor pattern produced the line designated ‘PH-2015-1074’ having a whitespotted flower color pattern with a red petal base color, as shown inFIG. 3.

As shown in FIG. 3, the spots vary from circular to irregular shape,vary in size, and are present on at least one petal of the flower.

4. Breeding of the Line ‘PH-2015-1065’ (FIG. 4)

Petunia line ‘PH-2015-1065’, which displays petals having a yellow-greenspotted flower color pattern in combination with a yellow-green starpattern on a red-purple base petal color, is the result of a plannedbreeding program. In 2012, PH-2012-3528′, which was the first seedlingdiscovered by Applicant showing the novel spotted flower color pattern,was crossed with a proprietary seedling designed ‘PH-2011-2416’, whichhad solid-red flowers. The resulting progeny from this cross,‘PH-2013-4407’ displayed purple flowers having a white spotted flowercolor pattern. ‘PH-2013-4407’ was further crossed with the solid blackflower colored commercial variety ‘Surprise Midnight Cowboy’(‘Duesurmidco’). Among the progeny of this cross, ‘PH-2014-0040’ wasselected which displayed dark-burgundy flowers with yellow spots.‘PH-2014-0040’ was further crossed with a proprietary Petunia seedlingdesignated PH-2014-1019′, which also had solid-black flowers, to producethe line designated ‘PH-2015-1065’, which has a yellow-green spottedflower color pattern in combination with a yellow-greenish star patternon a base red-purple petal color, as shown in FIG. 4.

As shown in FIG. 4, the spots vary from circular to irregular shape,vary in size, and are present on at least one petal of the flower.

5. Breeding of the Line ‘PH-2016-2150’ (FIG. 5)

Petunia line ‘PH-2016-2150’, as shown in FIG. 5, shows flowers having acream-yellow spotted flower color pattern in combination with acream-yellow star pattern on a dark violet base flower color. This photois an example of the spotted flower color pattern in combination with aborder pattern and a star pattern. and is the result of a plannedbreeding program. Petunia seedling ‘PH-2016-2150’ was selected inJanuary 2016 among progeny from a cross between two proprietaryseedlings, ‘PH-2015-1118’ and ‘PH-2015-1132’, both of which have flowerswith a cream-yellow spotted flower color pattern on a dark-violet basepetal color. ‘PH-2015-1118’ and ‘PH-2015-1132’ are derived from theprogeny of the third filial cross of the proprietary Petunia seedling‘PH-2012-3528’, which was the first seedling discovered by Applicantshowing the novel spotted flower color pattern, with proprietary andcommercial variety FAMOUS ‘Dark Blue’ (‘KLEPH08153’), which hassolid-blue flowers.

As shown in FIG. 5, the spots vary from circular to irregular shape,vary in size, and are present on at least one petal of the flower.

6. Breeding of the Line ‘PH-2015-1874’ (FIG. 6)

Petunia line ‘PH-2015-1874’, as shown in FIG. 6, showing white spots incombination with a white border on a blue base petal color. This photois an example of a flower with a solid border pattern in combinationwith the spotted flower color pattern of the present application, and isthe result of a planned breeding program. ‘PH-2015-1874’ was selected in2015 from a cross between the proprietary variety FAMOUS ‘Dark VioletPicotee’ (‘KLEPH14250’) and the proprietary Petunia variety ‘KLEPH15313’(also commercially known as NIGHT SKY Petunia and shown in FIGS. 1 and2). Whereas the female parent FAMOUS ‘Dark Violet Picotee’(‘KLEPH14250’) displays dark-violet flowers with a white border, theflowers of the male parent ‘KLEPH15313’, display a white spotted flowercolor pattern on a purple base flower color.

As shown in FIG. 6, the spots vary from circular to irregular shape,vary in size, and are present on at least one petal of the flower.

7. Breeding of the Line ‘PH-2016-2068’ (FIG. 7)

Petunia line ‘PH-2016-2068’, as shown in FIG. 7, displays flowers havinga white spotted flower color pattern on red star pattern in combinationwith a white base flower color and is the result of a planned breedingprogram. In 2012, PH-2012-3528′, which was the first seedling discoveredby Applicant showing the novel spotted flower color pattern, was crossedwith the proprietary Petunia seedling ‘PH-2011-2416’, which hadsolid-red flowers. The resulting progeny from this cross,‘PH-2013-4407’, displayed purple flowers with a white spotted flowercolor pattern. ‘PH-2013-4407’ was further crossed with the proprietaryPetunia seedling ‘PH-2013-4418’, which displayed blue flowers having awhite spotted flower color pattern. ‘PH-2013-4418’ resulted from a crossbetween ‘PH-2012-3528’ and the solid-orange-red colored varietyHEADLINER ‘Electric Orange’ (KLEPH14249′). Among the progeny of thecross between ‘PH-2013-4407 and ‘PH-2013-4418’, the inventors selectedthe seedling ‘PH-2014-0439’, which displayed red flowers having a whitespotted flower color pattern. A further cross of ‘PH-2014-0439’, withthe commercial Petunia variety ‘Viva Bright Red’ produced the variety‘PH-2016-2068’, which displays flowers having a white spotted flowercolor pattern on a red star pattern in combination with a white basepetal color.

As shown in FIG. 7, the spots vary from circular to irregular shape,vary in size, and are present on at least one petal of the flower.

8. Breeding of the Line ‘PH-2015-1080’ (FIG. 8)

Petunia line ‘PH-2015-1080’, as shown in FIG. 8, displays flowers havinga white spotted flower color pattern on a pink base petal color and isthe result of a planned breeding program. In 2012, PH-2012-3528′, whichwas the first seedling discovered by Applicant showing the novel spottedflower color pattern, was crossed with the proprietary Petunia seedling‘PH-2011-2416’, which had solid-red flowers. The resulting progeny fromthis cross, ‘PH-2013-4407’, displayed purple flowers having a whitespotted flower color pattern. ‘PH-2013-4407’ was further crossed withthe proprietary Petunia seedling ‘PH-2013-4418’ which displayed blueflowers having a white spotted flower color pattern. ‘PH-2013-4418’resulted from a cross between ‘PH-2012-3528’ and the solid-orange-redcolored variety HEADLINER ‘Electric Orange’ (‘KLEPH14249’). Among theprogeny of the cross between ‘PH-2013-4407’ and ‘PH-2013-4418’, theinventors selected the seedling ‘PH-2014-0443’, which displayeddark-pink flowers having a white spotted flower color pattern. A furthercross of ‘PH-2014-0443’ with the commercial Petunia variety STARLET‘Magenta’ (‘KLEPH15254’) produced the variety ‘PH-2015-1080’, whichshows flowers having a white spotted flower color pattern on a pink basepetal color.

As shown in FIG. 8, the spots vary from circular to irregular shape,vary in size, and are present on at least one petal of the flower.

9. Further Features of the Spotted Flower Color Pattern

The size and diameter of the spots may vary from 1 mm up to 1 cm indiameter and may measure anywhere in between. The shape may be round orirregular. Further, the border of the spots may be clear or indistinct.Any of these characteristics can be shown in FIGS. 1-8.

Furthermore, the spotted flower color pattern may appear in combinationwith other color patterns, e.g. star pattern, border pattern or centers(Please see FIGS. 4-7) or in combinations of more than two of thesepatterns.

The number of spots on the entire flower may vary up to several hundredspots. If the spotted flower color pattern is combined with anothercolor pattern, the number of respective spots may reduce accordingly tothe size of the respective spotted area. Petunia flower size wasmeasured for several-hundred progeny having the spotted flower colorpattern. Petunia flower sizes may vary from 2.5 cm to 10.5 cm indiameter.

Additionally, the novel spotted flower color pattern may appear onsingle flowers with five petals, which may or may not be fused, or,alternatively it may emerge on double-flowering inflorescences havingmore than five petals.

Furthermore, the spotted flower color pattern may emerge on plantshaving an upright habit, as well as on plants having a spreading habit.Further, the vigor of the Petunia plants showing the unique spottedflower color pattern may vary from very compact to very vigorous.

The Petunia plants with the unique color pattern can be used as potplants, container plants or in window boxes as well as in the field.

References to Known Patent Status of Referenced Petunia Varieties

The known patent status of the several above-referenced varieties are asfollows: ‘PH-2012-3528’, ‘PH-2015-1118’, ‘PH-2015-1132’, ‘PH-2011-2416’,‘PH-2013-4418’, ‘KLEPH08153’, ‘Duesurmidco’, and ‘KLEPH14250’ areunpatented; the patent status of ‘Can Can’ is unknown; the U.S. patentnumber of KLEPH15245 is USPP27,310; the U.S. patent number of KLEPH14249is USPP27,431; the U.S. patent number of KLEPH15313 is USPP27,237; andthe U.S. patent number of ‘Duesurmar’ is USPP21,673.

10. Molecular Background of the Spotted Flower Color Pattern in Petunia

The Petunia variety ‘Can Can’ as mentioned above exhibits a bicolorcolor pattern similar to other star-type Petunias. In this group (ofstar-type Petunia), it was shown that the pattern is induced bysequence-specific RNA degradation of Chalcone Synthase, the firstspecific enzyme in anthocyanin pigment biosynthesis. Please see Morita,Y., et al. “Tandemly arranged chalone synthase A genes contribute to thespatially regulated expression in siRNA and the natural bicolor floralphenotype in Petunia hybrida” The Plant Journal. 70:739-749 (2012).Sequence-specific RNA degradation occurs during a process calledpost-transcriptional gene silencing (PTGS) which protects the cell fromviral infection. PTGS is triggered by over threshold-levels of viralgene mRNA. Also, over threshold-amounts of plant gene mRNA can triggerPTGS. Please see Baulcombe, D. “RNA silencing in plants” Nature.431:356-363 (2004).

To prove whether PTGS is also responsible for the spotted flower colorpattern in the claimed spotted Petunia plants of the present applicationthat were derived from ‘Can Can’, ‘KLEPH15313’ was subjected toinfection with cucumber mosaic virus (CMV). This virus is known toencode a protein, which suppresses PTGS by preventing translocation ofthe silencing signal. Please see Baulcombe, D. “Viral suppression ofsystemic silencing” Trends Microbiol. 10:306-308 (2002). The result is astrong reduction or total loss of white petal areas on star-type Petuniaflowers. Please see Koseki, M., et al. “The star-type color pattern inPetunia hybrida ‘Red Star’ flowers is induced by sequence-specificdegradation of chalcone synthase RNA” Plant Cell Physiol. 46: 1879-1883(2005).

Three CMV isolates were selected for infection, because they causedsevere symptoms on Nicotiana benthamiana: isolate PV-0506 of subgroup IBlead to stunted plant growth, isolate PV-0474 of subgroup IB triggeredleaf deformations with shoestring leaves, isolate PV-0036 of subgroup IAinduced severe chlorosis on infected N. benthamiana plants. RNAs of thethree isolates were extracted, transcribed, amplified and successfullycloned into the pCB vector pDIVA3. With Agrobacterium tumefaciensbacteria, the cloned RNAs could be propagated and infiltrated in N.benthamiana plants. The cloned RNAs were part of the vector T-DNAs thatwere transferred by the bacteria into plant cells in which the viralgenes were transiently expressed. Crude leaf extracts containing the CMVparticles of one of the isolates each was used to inoculate Petuniaplants by carborundum treatment.

CMV Infection of Petunia Variety ‘KLEPH15313’

The three clones of PV-0506, PV-0474 and PV-0036 successfully inoculatedPetunia×hybrida plants of variety ‘KLEPH15313’. Twenty Petunia plants(four-weeks old) were inoculated per clone. As negative controls, 12Petunia plants were mock inoculated only with buffer and 12 Petuniaplants were left untreated. Plants were kept in greenhouse and symptomdevelopment was observed

Plants infected with clone PV-0506 were affected in growth and displayedsome chlorosis as well as necrosis. Plants and flowers were slightlysmaller than healthy plants and flowers. Additionally, most of theflowers were completely purple (FIG. 9 D) or had a purple smear in thewhite petal tips (FIG. 9 C). Some flowers had an unaltered appearance.Plants inoculated with clone PV-0474 looked healthy. In plant size andgrowth, they resembled the mock and untreated plants. Flower number andsize was also unchanged, but there were some changes in flowerappearance. While the plants produced mostly normal flower types (FIGS.9 A and B), there were a few purple flowers having white petal tips andpurple “smears” with vanishing white dots (FIG. 9C) that were produced,and some flowers were completely purple (FIG. 9D). The clone of isolatePV-0036 caused severe symptoms with necrosis and chlorosis of leavesproducing a dark green dot pattern on light green leaves (FIG. 9E).Plants and flowers were smaller in size and the majority of flowers werecompletely purple (FIG. 9D).

Comparative CMV Infection of Petunia Flowers with Different ColorPatterns

Three additional Petunia flower pattern types were infected with clonePV-0506 as described above: line PH-2014-0028′, exhibiting a starpattern-type flower, line ‘PH-2007-0103’, exhibiting a Picotee flowerpattern with solid white border pattern, and line PH-2015-1874′described above, which has a solid border pattern in combination withthe spotted flower color pattern. Six plants each were CMV-infected, and4 plants of each line served as a negative control. Plants werecultivated in a greenhouse and evaluated 4 weeks after mechanicalinfection as described above.

Infected plants and flowers of line ‘PH-2014-0028’ were smaller thanhealthy plants and flowers and showed some necrosis. Additionally, allflowers of this infected line lost the star-pattern flower and werecompletely purple (FIGS. 10 A3 and A4). This in accordance with earlierobservations on CMV-infected star-type flower pattern (Koseki, 2005) andvalidates this PTGS inhibition experiment. Similarly, infected plants ofline ‘PH-2007-0103’ showed retarded growth, but with only some necrosisand no chloroses. Plants and flowers were a bit smaller than healthyplants and flowers. But in contrast to line ‘PH-2014-0028’, flowersretained their white margin unaffected (FIGS. 10B3 and 10B4). Finally,line ‘PH-2015-187’ showed dwarfed plants and smaller flower size uponinfection. Flowers exhibited vanishing white dots or a total loss ofwhite dots, whereas the white margin remained unaffected (FIGS. 10C3 and10C4).

These experiments clearly document that the appearance of spotted flowercolor pattern originates from PTGS in single, stochastically (randomly)distributed cells and the spread of the PTGS signal to adjacent cells.When the translocation of the silencing signal is prevented by viralinteraction, the white spots or dots vanish until completedisappearance. The same mechanism has been proposed for star-patternPetunia (See Koseki et a. 2005), however the phenotype of white spots ordots was unexpected and not previously shown. The formation of a whiteflower margin as in Picotee line ‘PH-2007-0103’ originates from amolecular mechanism different from PTGS.

Expression Analysis of the Chalcone Synthase A Gene

To further investigate the molecular background of the spotted flowercolor pattern, a comparative expression analysis was performed on thefirst specific step in anthocyanin biosynthesis, the Chalcone Synthase Agene. This key gene has been shown before to be the target of PTGS inwhite petal areas (Koseki et al. 2005, Morita et al. 2012). For primergeneration, publicly available sequences of the Petunia×hybrida ChalconeSynthase A gene were aligned. The deduced consensus sequence resulted inPrimer CHSA-for (SEQ ID NO:1) and CHSA-rev (SEQ ID NO:2), respectively.Total RNA was isolated using INVITRAP® Spin Plant RNA Mini Kit (Stratek,Germany) according to the manufacturer's instructions. cDNA wassubsequently generated using the M-MLV Reverse Transcriptase Kit(Promega, USA) according to the manufacturer's instructions. ThePCR-Reaction for Actin or Chalcone Synthase A genes contained in a finalvolume reaction of 25 μL: 1 μL of cDNA as a template, 2 μL of 10×PCRBuffer, 1.2 μL of MgCl₂, 0.2 μL of dNTP mix (10 mM each), 0.5 μL of eachspecific primer (10 pmol) and 0.2 μL of Taq Polymerase. The cyclingconditions were an initial denaturation at 95° C. for 3 minutes; 30cycles of 95° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 30seconds; and a final extension at 72° C. for 10 minutes. PCR productswere visualized in a 2% (w/v) agaroses gel, at 120 W for 90 minutes.

FIG. 11 shows the results, that Chalcone Synthase A (CHS-A) expressioncannot be observed in white petal tissue of variety ‘KLEPH15313’,whereas it can clearly be detected in white petal tissue of line‘PH-2007-0103’ (FIG. 11). This supports the hypothesis that the whitespots or dots and areas in ‘KLEPH15313’ originate from single,stochastically (randomly) distributed petal cells in which ChalconeSynthase, the first specific step in anthocyanin biosynthesis, has beenknocked down by PTGS. The white spots or dots become visible bysubsequent spread of the silencing signal to adjacent petal cells andtheir final size is determined by the extent of this spread. Thismolecular mechanism is unexpected and different from the Picotee-typePetunia flowers, where the white margin area tissue is not influenced byPTGS and exhibits Chalcone Synthase A activity (See FIG. 11).

11. Intergeneric Breeding of the Petunia Plants Having the SpottedFlower Color Pattern with Calibrachoa

Petunia plants herein exhibiting the spotted flower color pattern may bebred with Calibrachoa plants to produce Petunia-Calibrachoa hybridplants exhibiting the spotted flower color pattern. Such intergenericbreeding is well-known in the art.

FURTHER EMBODIMENTS

Breeding with Petunia

The goal of ornamental plant breeding is to develop new, unique andsuperior ornamental varieties and hybrids. The breeder initially selectsand crosses two or more parental lines, followed by repeated crossingand selfing, producing many new genetic combinations. The breeder cantheoretically generate billions of different genetic combinations viacrossing, selection, selfing and mutations. Therefore, a breeder willnever develop the same variety genetically and having the same traitsfrom the exact same parents.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions and further selections arethen made during and at the end of the growing season. The varietiesthat are developed are unpredictable because the breeder's selectionoccurs in unique environments with no control at the DNA level, and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same variety twice by using the sameoriginal parents and the same selection techniques. Thisunpredictability results in the expenditure of large amounts of researchmonies to develop superior new Petunia varieties.

Breeding programs combine desirable traits from two or more varieties orvarious broad-based sources into breeding pools from which varieties aredeveloped by further crossing or selfing and selection of desiredphenotypes. Pedigree breeding is used commonly for the improvement ofself-pollinating plants. Two parents that possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁s. Selection of the bestindividuals may begin in the F₂ population; then, beginning in the F₃,the best individuals in the best families are selected. Replicatedtesting of families can begin in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new varieties.

Using Petunia Plants Having a Spotted Flower Color Pattern to DevelopOther Petunia Plants Having a Spotted Flower Color Pattern

The Petunia plants herein can also provide a source of breeding materialthat may be used to develop new Petunia plants and varieties. Plantbreeding techniques known in the art and used in a Petunia plantbreeding program include, but are not limited to, recurrent selection,mass selection, bulk selection, hybridization, mass selection,backcrossing, pedigree breeding, open-pollination breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection, making double haploids, mutagenesis and transformation. Oftencombinations of these techniques are used. There are many analyticalmethods available to evaluate a new variety. The oldest and mosttraditional method of analysis is the observation of phenotypic traits,but genotypic analysis may also be used.

Additional Breeding Methods

Any plants produced using the Petunia plants disclosed in the presentapplication as at least one parent are also an embodiment. These methodsare well-known in the art and some of the more commonly used breedingmethods are described herein. Descriptions of breeding methods can befound in one of several reference books (e.g., Allard, “Principles ofPlant Breeding” (1999); Vainstein, “Breeding for Ornamentals: Classicaland Molecular Approaches,” Kluwer Academic Publishers (2002); Callaway,“Breeding Ornamental Plants,” Timber Press (2000); and Bragdø, Marie,“Inter-specific Crosses in Lupinus: Cytology and Inheritance of FlowerColor,” Institute of Genetics and Plant Breeding, Agricultural Collegeof Norway, Vollebekk, Norway (Sep. 28, 1956).

Breeding steps that may be used in the Petunia plant breeding programcan include for example, pedigree breeding, backcrossing, mutationbreeding, and recurrent selection. In conjunction with these steps,techniques such as RFLP-enhanced selection, genetic marker enhancedselection (for example, SSR markers), and the making of double haploidsmay be utilized.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which Petunia plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as pollen, ovules, embryos,protoplasts, meristematic cells, callus, leaves, anthers, cotyledons,hypocotyl, pistils, roots, root tips, seeds, flowers, petiole, shoot, orstems and the like.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such asPetunia spotted flower color pattern phenotype and another differentPetunia having one or more desirable characteristics that is lacking orwhich complements the Petunia spotted flower color pattern phenotype. Ifthe two original parents do not provide all the desired characteristics,other sources can be included in the breeding population. In thepedigree method, superior plants are selfed and selected in successivefilial generations. In the succeeding filial generations, theheterozygous condition gives way to homogeneous varieties as a result ofself-pollination and selection. Typically in the pedigree method ofbreeding, five or more successive filial generations of selfing andselection is practiced: F₁ to F₂; F₂ to F₃; F₃ to F₄; F₄ to F₅; etc.After a sufficient amount of inbreeding, successive filial generationswill serve to increase seed of the developed variety. Preferably, thedeveloped variety comprises homozygous alleles at about 95% or more ofits loci.

Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous variety orinbred line which is the recurrent parent. The source of the trait to betransferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have the attributes of the recurrentparent and the desirable trait transferred from the donor parent.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety, the donor parent, to adeveloped variety called the recurrent parent, which has overall goodcommercial characteristics and yet lacks that desirable trait or traits.However, the same procedure can be used to move the progeny toward thegenotype of the recurrent parent, but at the same time retain manycomponents of the nonrecurrent parent by stopping the backcrossing at anearly stage and proceeding with selfing and selection. For example, aPetunia plant may be crossed with another variety to produce afirst-generation progeny plant. The first-generation progeny plant maythen be backcrossed to one of its parent varieties to create a BC₁ orBC₂. Progeny are selfed and selected so that the newly developed varietyhas many of the attributes of the recurrent parent and yet several ofthe desired attributes of the nonrecurrent parent. This approachleverages the value and strengths of the recurrent parent for use in newPetunia varieties.

Therefore, another embodiment is a method of making a backcrossconversion of the Petunia having the spotted flower color pattern,comprising the steps of crossing a Petunia plant having the spottedflower color pattern phenotype with a donor plant comprising a desiredtrait, selecting an F₁ progeny plant comprising the desired trait, andbackcrossing the selected F₁ progeny plant to a plant of Petunia havingthe spotted flower color pattern phenotype. This method may furthercomprise the step of obtaining a molecular marker profile of Petuniahaving the spotted flower color pattern phenotype and using themolecular marker profile to select for a progeny plant with the desiredtrait and the molecular marker profile of Petunia having the spottedflower color pattern phenotype.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. Petunia having the spotted flower colorpattern phenotype are suitable for use in a recurrent selection program.The method entails individual plants cross-pollinating with each otherto form progeny. The progenies are grown and the superior progeniesselected by any number of selection methods, which include individualplant, half-sib progeny, full-sib progeny, and selfed progeny. Theselected progenies are cross-pollinated with each other to form progenyfor another population. This population is planted and again superiorplants are selected to cross-pollinate with each other. Recurrentselection is a cyclical process and therefore can be repeated as manytimes as desired. The objective of recurrent selection is to improve thetraits of a population. The improved population can then be used as asource of breeding material to obtain new varieties for commercial orbreeding use, including the production of a synthetic variety. Asynthetic variety is the resultant progeny formed by the intercrossingof several selected varieties.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection, seeds fromindividuals are selected based on phenotype or genotype. These selectedseeds are then bulked and used to grow the next generation. Bulkselection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk, andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Also, instead of self-pollination, directed pollinationcould be used as part of the breeding program.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating plants. A genetically variablepopulation of heterozygous individuals is either identified, or created,by intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Mutation Breeding

Mutation breeding is another method of introducing new traits intoPetunia having the spotted flower color pattern phenotype. Mutationsthat occur spontaneously or are artificially induced can be usefulsources of variability for a plant breeder. The goal of artificialmutagenesis is to increase the rate of mutation for a desiredcharacteristic. Mutation rates can be increased by many different meansincluding temperature, long-term seed storage, tissue cultureconditions, radiation; such as X-rays, Gamma rays (e.g., cobalt 60 orcesium 137), neutrons, (product of nuclear fission by uranium 235 in anatomic reactor), Beta radiation (emitted from radioisotopes such asphosphorus 32 or carbon 14), or ultraviolet radiation (preferably from2500 to 2900 nm), or chemical mutagens (such as base analogues(5-bromo-uracil)), related compounds (8-ethoxy caffeine), antibiotics(streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards,epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones),azide, hydroxylamine, nitrous acid, or acridines. Once a desired traitis observed through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in Vainstein, “Breeding for Ornamentals:Classical and Molecular Approaches,” Kluwer Academic Publishers (2002).In addition, mutations created in other Petunia plants may be used toproduce a backcross conversion of Petunia having the spotted flowercolor pattern phenotype that comprises such mutation.

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformant plants obtained with the protoplasm of the subject Petuniaplants are intended to be within the scope of the embodiments of theapplication.

Single-Gene Conversions

When the term Petunia plant is used in the context of an embodiment ofthe present application, this also includes any single gene conversionsof Petunia having the spotted flower color pattern phenotype. The termsingle gene converted plant as used herein refers to those Petuniaplants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition tothe single gene transferred into the variety via the backcrossingtechnique. Backcrossing methods can be used with one embodiment of thepresent application to improve or introduce a characteristic into thevariety. The term “backcrossing” as used herein refers to the repeatedcrossing of a hybrid progeny back to the recurrent parent, i.e.,backcrossing 1, 2, 3, 4, 5, 6, 7, 8, or more times to the recurrentparent. The parental Petunia plant that contributes the gene for thedesired characteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental Petunia plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman & Sleper(1994). In a typical backcross protocol, the original variety ofinterest (recurrent parent) is crossed to a second variety (nonrecurrentparent) that carries the single gene of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a Petunia plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred gene from thenonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially important trait or traitsto the plant. The exact backcrossing protocol will depend on thecharacteristic or trait being altered to determine an appropriatetesting protocol. Although backcrossing methods are simplified when thecharacteristic being transferred is a dominant allele, a recessiveallele may also be transferred. In this instance it may be necessary tointroduce a test of the progeny to determine if the desiredcharacteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. These traits are well-known in theart.

Introduction of a New Trait or Locus into Petunia Having the SpottedFlower Color Pattern Phenotype

Petunia having the spotted flower color pattern phenotype represent anew base of genetics into which a new locus or trait may beintrogressed. Direct transformation and backcrossing represent twoimportant methods that can be used to accomplish such an introgression.The term backcross conversion and single locus conversion are usedinterchangeably to designate the product of a backcrossing program.

Backcross Conversions of Petunia Having the Spotted Flower Color PatternPhenotype

A backcross conversion of Petunia having the spotted flower colorpattern phenotype occurs when DNA sequences are introduced throughbackcrossing (Allard, “Principles of Plant Breeding” (1999) with Petuniahaving the spotted flower color pattern phenotype utilized as therecurrent parent. Both naturally occurring and transgenic DNA sequencesmay be introduced through backcrossing techniques. A backcrossconversion may produce a plant with a trait or locus conversion in atleast two or more backcrosses, including at least 2 crosses, at least 3crosses, at least 4 crosses, at least 5 crosses, and the like. Molecularmarker assisted breeding or selection may be utilized to reduce thenumber of backcrosses necessary to achieve the backcross conversion. Forexample, see, Openshaw, S. J., et al., Marker-assisted Selection inBackcross Breeding, Proceedings Symposium of the Analysis of MolecularData, Crop Science Society of America, Corvallis, Oreg. (August 1994),where it is demonstrated that a backcross conversion can be made in asfew as two backcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes ascompared to unlinked genes), the level of expression of the trait, thetype of inheritance (cytoplasmic or nuclear), and the types of parentsincluded in the cross. It is understood by those of ordinary skill inthe art that for single gene traits that are relatively easy toclassify, the backcross method is effective and relatively easy tomanage. See, Allard, “Principles of Plant Breeding” (1999). Desiredtraits that may be transferred through backcross conversion include, butare not limited to, sterility (nuclear and cytoplasmic), fertilityrestoration, drought tolerance, nitrogen utilization, ornamentalfeatures, disease resistance (bacterial, fungal, or viral), insectresistance, and herbicide resistance. In addition, an introgression siteitself, such as an FRT site, Lox site, or other site specificintegration site, may be inserted by backcrossing and utilized fordirect insertion of one or more genes of interest into a specific plantvariety. In some embodiments, the number of loci that may be backcrossedinto Petunia having the spotted flower color pattern phenotype is atleast 1, 2, 3, 4, or 5, and/or no more than 6, 5, 4, 3, or 2. A singlelocus may contain several transgenes, such as a transgene for diseaseresistance that, in the same expression vector, also contains atransgene for herbicide resistance. The gene for herbicide resistancemay be used as a selectable marker and/or as a phenotypic trait. Asingle locus conversion of site specific integration system allows forthe integration of multiple genes at the converted loci.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes or genes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele requires growing and selfing thefirst backcross generation to determine which plants carry the recessivealleles. Recessive traits may require additional progeny testing insuccessive backcross generations to determine the presence of the locusof interest. The last backcross generation is usually selfed to givepure breeding progeny for the gene(s) being transferred, although abackcross conversion with a stably introgressed trait may also bemaintained by further backcrossing to the recurrent parent withselection for the converted trait.

Molecular Techniques Using Petunia Having the Spotted Flower ColorPattern Phenotype

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions. Traditional plant breeding has principally been the source ofnew germplasm, however, advances in molecular technologies have allowedbreeders to provide varieties with novel and much wanted commercialattributes. Molecular techniques such as transformation are popular inbreeding ornamental plants and well-known in the art. See Vainstein,“Breeding for Ornamentals: Classical and Molecular Approaches,” KluwerAcademic Publishers (2002).

Breeding with Molecular Markers

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs), and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing Petunia plants having the spotted flowercolor pattern phenotype. See Vainstein, “Breeding for Ornamentals:Classical and Molecular Approaches,” Kluwer Academic Publishers (2002).

Genetic Marker Profile Through SSR and First Generation Progeny

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile which can identify plants of the same variety, ora related variety, or be used to determine or validate a pedigree.Genetic marker profiles can be obtained by techniques such asRestriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) (which are also referred to asMicrosatellites), and Single Nucleotide Polymorphisms (SNPs), all ofwhich are well-known in the art.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of ornamental plants andPetunia and regeneration of plants therefrom is well-known and widelypublished. For example, reference may be had to do Valla Rego, Lucianaet al., Crop Breeding and Applied Technology. 1(3): 283-300 (2001);Komatsuda, T., et al., Crop Sci., 31:333-337 (1991); Stephens, P. A., etal., Theor. Appl. Genet., 82:633-635 (1991); Komatsuda, T., et al.,Plant Cell, Tissue and Organ Culture, 28:103-113 (1992); Dhir, S., etal., Plant Cell Reports, 11:285-289 (1992); Pandey, P., et al., Japan J.Breed., 42:1-5 (1992); and Shetty, K., et al., Plant Science, 81:245-251(1992). Thus, another embodiment is to provide cells which upon growthand differentiation produce Petunia plants having the physiological andmorphological characteristics of Petunia described in the presentapplication.

Regeneration refers to the development of a plant from tissue culture.The term “tissue culture” indicates a composition comprising isolatedcells of the same or a different type or a collection of such cellsorganized into parts of a plant. Exemplary types of tissue cultures areprotoplasts, calli, plant clumps, and plant cells that can generatetissue culture that are intact in plants or parts of plants, such aspollen, ovules, embryos, protoplasts, meristematic cells, callus,leaves, anthers, cotyledons, hypocotyl, pistils, roots, root tips,flowers, seeds, petiole, shoot, or stems, and the like. Means forpreparing and maintaining plant tissue culture are well-known in theart.

Targeted Gene Editing

Targeted gene editing can be done using CRISPR/Cas9 technology (Saunders& Joung, Nature Biotechnology, 32, 347-355, 2014). CRISPR is a type ofgenome editing system that stands for Clustered Regularly InterspacedShort Palindromic Repeats. This system and CRISPR-associated (Cas) genesenable organisms, such as select bacteria and archaea, to respond to andeliminate invading genetic material. Ishino, Y., et al. J. Bacteriol.169, 5429-5433 (1987). These repeats were known as early as the 1980s inE. coli, but Barrangou and colleagues demonstrated that S. thermophiluscan acquire resistance against a bacteriophage by integrating a fragmentof a genome of an infectious virus into its CRISPR locus. Barrangou, R.,et al. Science 315, 1709-1712 (2007). Many plants have already beenmodified using the CRISPR system. See for example, U.S. ApplicationPublication No. WO2014068346 (György et al., Identification of aXanthomonas euvesicatoria resistance gene from pepper (Capsicum annuum)and method for generating plants with resistance), Martinelli, F. etal., “Proposal of a Genome Editing System for Genetic Resistance toTomato Spotted Wilt Virus” American Journal of Applied Sciences 2014,and Noman, A. et al., “CRISPR-Cas9: Tool for Qualitative andQuantitative Plant Genome Editing” Frontiers in Plant Science Vol. 7Nov. 2016.

Gene editing can also be done using crRNA-guided surveillance systemsfor gene editing. Additional information about crRNA-guided surveillancecomplex systems for gene editing can be found in the followingdocuments, which are incorporated by reference in their entirety: U.S.Application Publication No. 2010/0076057 (Sontheimer et al., Target DNAInterference with crRNA); U.S. Application Publication No. 2014/0179006(Feng, CRISPR-CAS Component Systems, Methods, and Compositions forSequence Manipulation); U.S. Application Publication No. 2014/0294773(Brouns et al., Modified Cascade Ribonucleoproteins and Uses Thereof);Sorek et al., Annu. Rev. Biochem. 82:273-266, 2013; and Wang, S. et al.,Plant Cell Rep (2015) 34: 1473-1476. Therefore, it is another embodimentto use the CRISPR system on the Petunia plants of the instantapplication to for example, modify traits and tolerances to pests andviruses.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

One or more aspects may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the embodiments is, therefore,indicated by the appended claims rather than by the foregoingdescription. All changes which come within the meaning and range ofequivalency of the claims are to be embraced within their scope. Theforegoing discussion of the embodiments has been presented for purposesof illustration and description. The foregoing is not intended to limitthe embodiments to the form or forms disclosed herein. In the foregoingFurther Embodiments, for example, various features of the embodimentsare grouped together in one or more embodiments for the purpose ofstreamlining the disclosure. This method of disclosure is not to beinterpreted as reflecting an intention that the claimed embodimentsrequire more features than are expressly recited in each claim. Rather,as the following claims reflect, inventive aspects lie in less than allfeatures of a single foregoing disclosed embodiment. Thus, the followingclaims are hereby incorporated into this Further Embodiments, with eachclaim standing on its own as a separate preferred embodiment.

Moreover, though the description of the embodiments has includeddescription of one or more embodiments and certain variations andmodifications, other variations and modifications are within the scopeof the embodiments (e.g., as may be within the skill and knowledge ofthose in the art, after understanding the present disclosure). It isintended to obtain rights which include alternative embodiments to theextent permitted, including alternate, interchangeable and/or equivalentstructures, functions, ranges or acts to those claimed, whether or notsuch alternate, interchangeable and/or equivalent structures, functions,ranges or acts are disclosed herein, and without intending to publiclydedicate any patentable subject matter.

The use of the terms “a,” “an,” and “the,” and similar referents in thecontext of describing the embodiments (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the embodiments and does not pose a limitation on the scopeof the embodiments unless otherwise claimed. No language in thespecification should be construed as indicating any non-claimed elementas essential to the practice one or more embodiments.

DEPOSIT INFORMATION

A representative sample of proprietary Petunia seed of the Klemm+SohnGmbH & Co. KG, which is a mixture of the lines disclosed in theapplication, comprising a dominant allele with incomplete penetrancethat produces a spotted flower color pattern, and wherein said Petuniaplants grown from said seed express a spotted flower color pattern, wasmade with the National Collections of Industrial, Food and MarineBacteria (NCIMB), Ferguson Building, Craibstone Estate, Bucksburn,Aberdeen, Scotland, AB21 9YA, United Kingdom. The date of deposit of2,000 seeds was Mar. 23, 2016 and the NCIMB No. is 42563. On Jan. 19,2017, an additional deposit of 500 seeds was made with NCIMB. Thedeposit of 2,500 seeds wherein said Petunia plants express a spottedflower color pattern was taken from the same deposit maintained byKlemm+Sohn GmbH & Co. KG since prior to the filing date of thisapplication. Upon issuance of a patent, all restrictions on theavailability to the public of the deposit will be irrevocably removedconsistent with all of the requirements of 37 C.F.R. § § 1.801-1.809.The deposit will be maintained in the depository for a period of thirtyyears, or five years after the last request, or for the enforceable lifeof the patent, whichever is longer, and will be replaced as necessaryduring the period.

What is claimed is:
 1. A Petunia plant comprising a dominant allele withincomplete penetrance that produces a spotted flower color pattern,wherein said pattern is produced from a complete post-transcriptionalgene silencing of the Chalcone Synthase A gene in single randomlydistributed cells of the petals, and wherein said spotted flower colorpattern comprises a Petunia plant having one or more spots on each petalof every flower, wherein said Petunia plant is produced from seeddeposited under NCIMB Accession Number
 42563. 2. A Petunia seed producedby growing the plant of claim
 1. 3. A method of producing a Petuniaplant, or a plant part thereof, by growing the seed of claim
 2. 4. Aplant part of the plant of claim 1, wherein the plant part comprises acell, seed, protoplast, tissue culture, or vegetative cutting.
 5. Atissue culture produced from tissue, callus, cells, or protoplasts fromthe plant of claim 1, wherein said tissue, callus, cells or protoplastsare produced from a plant part selected from the group consisting ofpollen, ovules, embryos, meristematic cells, callus, leaves, anthers,cotyledons, hypocotyl, pistils, roots, root tips, flowers, seeds,petiole, and stems.
 6. A Petunia plant regenerated from the tissueculture of claim
 5. 7. A method for producing Petunia seed, said methodcomprising crossing two Petunia plants and harvesting the resultantPetunia seed, wherein at least one Petunia plant is the Petunia plant ofclaim
 1. 8. The method of claim 7, further comprising growing said seedand selecting a plant having the spotted flower color pattern.
 9. ThePetunia plant of claim 1, wherein said spot is white, cream, yellow,yellow-green, or combinations thereof in color.
 10. The Petunia plant ofclaim 1, wherein the flower of said Petunia is yellow, orange, red,brown, blue, black, pink, violet, or combinations and shades thereof.11. The Petunia plant of claim 1, wherein said spot is shaped round orirregular.
 12. The Petunia plant of claim 1, wherein said flower is asingle flower.
 13. The Petunia plant of claim 1, wherein said flower isa double flower.
 14. The Petunia plant of claim 1, wherein the spottedflower color pattern is present in combination with a star pattern, aborder pattern, or a star and a border pattern.
 15. A method ofvegetatively propagating the Petunia plant of claim 1 comprising thesteps of: (a) collecting tissue capable of being propagated from theplant according to claim 1; (b) cultivating said tissue to obtainproliferated shoots; and (c) rooting said proliferated shoots to obtainrooted plantlets.
 16. A Petunia plant produced by the method of claim15.